Gram-positive microorganisms with an inactivated cysteine protease-2

ABSTRACT

The present invention relates to the identification of novel cysteine proteases in Gram-positive microorganisms. The present invention provides the nuclei acid and amino acid sequences for the  Bacillus subtilis  cysteine proteases CP1, CP2 and CP3. The present invention also provides host cells having a mutation or deletion of part or all of the gene encoding CP1, CP2 or CP3. The present invention also provides host cells further comprising nucleic acid encoding desired heterologous proteins such as enzymes. The present invention also provides a cleaning composition comprising a cysteine protease of the present invention.

FIELD OF THE INVENTION

[0001] The present invention relates to cysteine proteases derived fromgram-positive microorganisms. The present invention provides nucleicacid and amino acid sequences of cysteine protease 1, 2 and 3 identifiedin Bacillus. The present invention also provides methods for theproduction of cysteine protease 1, 2 and 3 in host cells as well as theproduction of heterologous proteins in a host cell having a mutation ordeletion of part or all of at least one of the cysteine proteases of thepresent invention.

BACKGROUND OF THE INVENTION

[0002] Gram-positive microorganisms, such as members of the groupBacillus, have been used for large-scale industrial fermentation due, inpart, to their ability to secrete their fermentation products into theculture media. In gram-positive bacteria, secreted proteins are exportedacross a cell membrane and a cell wall, and then are subsequentlyreleased into the external media usually maintaining their nativeconformation.

[0003] Various gram-positive microorganisms are known to secreteextracellular and/or intracellular protease at some stage in their lifecycles. Many proteases are produced in large quantities for industrialpurposes. A negative aspect of the presence of proteases ingram-positive organisms is their contribution to the overall degradationof secreted heterologous or foreign proteins.

[0004] The classification of proteases found in microorganisms is basedon their catalytic mechanism which results in four groups: the serineproteases; metalloproteases; cysteine proteases; and aspartic proteases.These categories can be distinguished by their sensitivity to variousinhibitors. For example, the serine proteases are inhibited byphenylmethylsulfonylfluoride (PMSF) and diisopropylfluorophosphate(DIFP); the metalloproteases by chelating agents; the cysteine enzymesby iodoacetamide and heavy metals and the aspartic proteases bypepstatin. The serine proteases have alkaline pH optima, themetalloproteases are optimally active around neutrality, and thecysteine and aspartic enzymes have acidic pH optima (BiotechnologyHandbooks, Bacillus. vol. 2, edited by Harwood, 1989 Plenum Press,N.Y.).

[0005] The activity of cysteine protease depends on a catalytic dyad ofcysteine and histidine with the order differing between families. Thebest known family of cysteine proteases is that of papain havingcatalytic residues Cys-25 and His-159. Cysteine proteases of the papainfamily catalyze the hydrolysis of peptide, amide, ester, thiol ester andthiono ester bonds. Naturally occurring inhibitors of cysteine proteasesof the papain family are those of the cystatin family (Methods inEnzymology, vol. 244, Academic Press, Inc. 1994).

SUMMARY OF THE INVENTION

[0006] The present invention relates to the unexpected and surprisingdiscovery of three heretofore unknown or unrecognized cysteine proteasesfound in Bacillus subtilis, designated herein as CP1, CP2 and CP3 havingthe nucleic acid and amino acid as shown in FIGS. 1A-1B, FIGS. 5A-5B and6A-6B, respectively. The present invention is based, in part, upon thepresence of the characteristic cysteine protease amino acid motif GXCWAFfound in uncharacterised translated genomic nucleic acid sequences ofBacillus subtilis. The present invention is also based. in part upon thestructural relatedness that CP1 has with the cysteine protease papainspecifically with respect to the location of the catalytichistidine/alanine and asparagine/serine residues and the structuralrelatedness that CP1 has with CP2 and CP3.

[0007] The present invention provides isolated polynucleotide and aminoacid sequences for CP1, CP2 and CP3. Due to the degeneracy of thegenetic code, the present invention encompasses any nucleic acidsequence that encodes the CP1, CP2 and CP3 amino acid sequence shown inthe Figures.

[0008] The present invention encompasses amino acid variations of B.subtilis CP1, CP2 and CP3 amino acids disclosed herein that haveproteolytic activity. B. subtilis CP1, CP2 and CP3 as well asproteolytically active amino acid variations, thereof have applicationin cleaning compositions. The present invention also encompasses aminoacid variations or derivatives of CP1, CP2, CP3 that do not have thecharacteristic proteolytic activity as long as the nucleic acidsequences encoding such variations or derivatives would have sufficient5′ and 3′ coding regions to be capable of being integrated into agram-positive organism genome. Such variants would have applications ingram-positive expression systems where it is desirable to delete,mutate, alter or otherwise incapacitate the naturally occurring cysteineprotease in order to diminish or delete its proteolytic activity. Suchan expression system would have the advantage of allowing for greateryields of recombinant heterologous proteins or polypeptides.

[0009] The present invention provides methods for detecting grampositive microorganism homologs of B. subtilis CP1, CP2 and CP3 thatcomprises hybridizing part or all of the nucleic acid encoding B.subtilis CP1, CP2 and CP3 with nucleic acid derived from gram-positiveorganisms, either of genomic or cDNA origin. In one embodiment, thegram-positive microorganism is selected from the group consisting of B.licheniformis, B. lentus, B. brevis, B. stearothermophilus, B.alkalophilus, B. amyloliquefaciens, B. coagulans, B. circulans, B.lautus and Bacillus thuringiensis.

[0010] In yet another aspect, the present invention provides agram-positive microorganism having a mutation or deletion of part or allof the gene encoding CP1 and/or CP2 and/or CP3, which results in theinactivation of the CP1 and/or CP2 and/or CP3 proteolytic activity,either alone or in combination with mutations in other proteases, suchas apr, npr, epr, mpr for example, or other proteases known to those ofskill in the art. In one embodiment of the present invention, thegram-positive organism is a member of the genus Bacillus. In anotherembodiment, the Bacillus is Bacillus subtilis.

[0011] The production of desired heterologous proteins or polypeptidesin gram-positive microorganisms may be hindered by the presence of oneor more proteases which degrade the produced heterologous protein orpolypeptide. One advantage of the present invention is that it providesmethods and expression systems which can be used to prevent thatdegradation, thereby enhancing yields of the desired heterologousprotein or polypeptide. In another aspect, the gram-positive host havingone or more cysteine protease deletions is further geneticallyengineered to produce a desired protein.

[0012] In one embodiment of the present invention, the desired proteinis heterologous to the gram-positive host cell. In another embodiment,the desired protein is homologous to the host cell. The presentinvention encompasses a gram-positive host cell having a deletion orinterruption of the nucleic acid encoding the naturally occurringhomologous protein, such as a protease, and having nucleic acid encodingthe homologous protein re-introduced in a recombinant form. In anotherembodiment, the host cell produces the homologous protein. Accordingly,the present invention also provides methods and expression systems forreducing degradation of heterologous proteins produced in gram-positivemicroorganisms. The gram-positive microorganism may be normallysporulating or non-sporulating.

[0013] In a further aspect of the present invention, gram-positive CP1,CP2 or CP3 is produced on an industrial fermentation scale in amicrobial host expression system. In another aspect, isolated andpurified recombinant CP1, CP2 or CP3 is used in compositions of matterintended for cleaning purposes, such as detergents. Accordingly, thepresent invention provides a cleaning composition comprises one or moreof a gram-positive cysteine protease selected from the group consistingof CP1, CP2 and CP3. The cysteine protease may be used alone or incombination with other enzymes and/or mediators or enhancers.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] FIGS. 1A-1B shows the DNA and amino acid sequence for CP1 (YJDE).

[0015]FIG. 2 shows an amino acid alignment with papain (accession numberpapa_carpa.p) with the cysteine protease CP1, designated YJDE. For FIGS.2, 3 and 4, the motif GXCWAF has been marked along with the catalyticcysteine and the conserved catalytic histidine/alanine andasparagine/serine residues.

[0016]FIG. 3 shows amino acid alignment of CP1 (YJDE) with CP3 (PMI).

[0017]FIG. 4 shows the amino acid alignment of CP1 (YJDE) with CP2(YdhS).

[0018]FIG. 5A-5B shows the amino acid and nucleic acid sequence for CP2(YdhS).

[0019]FIG. 6A-6B shows the amino acid and nucleic acid sequence for CP3(PMI).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] Definitions

[0021] As used herein, the genus Bacillus includes all members known tothose of skill in the art, including but not limited to B. subtilis, B.licheniformis, B. lentus, B. brevis, B. stearothermophilus, B.alkalophilus, B. amyloliquefaciens, B. coagulans, B. ciculans, B. lautusand B. thuringiensis.

[0022] The present invention encompasses novel CP1, CP2 and CP3 fromgram positive organisms. In a preferred embodiment, the gram-positiveorganisms is a Bacillus. In another preferred embodiment, thegram-positive organism is Bacillus subtilis. As used herein, “B.subtilisCP1, CP2 or CP3” refers to the amino acid sequences shown in Figures.FIGS. 1A-1B show the amino acid and nucleic acid seqeunce for CP1(YJDE); FIGS. 5A-5B show the amino acid and nucleic acid sequence forCP2 (YDHS); and FIGS. 6A-6B show the amino acid and nucleic acidsequences for CP3 (PMI). The present invention encompasses amino acidvariations of the amino acid sequences disclosed in FIGS. 1A-1B and5A-5B and 6A-6B that have proteolytic activity. Such proteolytic aminoacid variants can be used in cleaning compositions. The presentinvention also encompasses B. subtilis amino acid variations orderivatives that are not proteolytically active. DNA encoding suchvariants can be used in methods designed to delete or mutate thenaturally occurring host cell CP1, CP or CP3.

[0023] As used herein, “nucleic acid” refers to a nucleotide orpolynucleotide sequence, and fragments or portions thereof, and to DNAor RNA of genomic or synthetic origin which may be double-stranded orsingle-stranded, whether representing the sense or antisense strand. Asused herein “amino acid” refers to peptide or protein sequences orportions thereof. A “polynucleotide homolog” as used herein refers to agram-positive microorganism polynucleotide that has at least 80%, atleast 90% and at least 95% identity to B.subtilis CP1, CP2 or CP3, orwhich is capable of hybridizing to B. subtilis CP1, CP2 or CP3 underconditions of high stringency and which encodes an amino acid sequencehaving cysteine protease activity.

[0024] The terms “isolated” or “purified” as used herein refer to anucleic acid or amino acid that is removed from at least one componentwith which it is naturally associated.

[0025] As used herein, the term “heterologous protein” refers to aprotein or polypeptide that does not naturally occur in a gram-positivehost cell. Examples of heterologous proteins include enzymes such ashydrolases including proteases, cellulases, amylases, carbohydrases, andlipases; isomerases such as racemases, epimerases, tautomerases, ormutases; transferases, kinases and phophatases. The heterologous genemay encode therapeutically significant proteins or peptides, such asgrowth factors, cytokines, ligands, receptors and inhibitors, as well asvaccines and antibodies. The gene may encode commercially importantindustrial proteins or peptides, such as proteases, carbohydrases suchas amylases and glucoamylases, cellulases, oxidases and lipases. Thegene of interest may be a naturally occurring gene, a mutated gene or asynthetic gene.

[0026] The term “homologous protein” refers to a protein or polypeptidenative or naturally occurring in a gram-positive host cell. Theinvention includes host cells producing the homologous protein viarecombinant DNA technology. The present invention encompasses agram-positive host cell having a deletion or interruption of the nucleicacid encoding the naturally occurring homologous protein, such as aprotease, and having nucleic acid encoding the homologous proteinre-introduced in a recombinant form. In another embodiment, the hostcell produces the homologous protein.

[0027] As used herein, the term “overexpressing” when refering to theproduction of a protein in a host cell means that the protein isproduced in greater amounts than its production in its naturallyoccurring environment.

[0028] As used herein, the phrase “proteolytic activity” refers to aprotein that is able to hydrolyze a peptide bond. Enzymes havingproteolytic activity are described in Enzyme Nomenclature, 1992, editedWebb Academic Press, Inc.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] The unexpected discovery of the cysteine proteases CP1, CP2 andCP3 in B. subtilis provides a basis for producing host cells, expressionmethods and systems which can be used to prevent the degradation ofrecombinantly produced heterologous proteins. In a preferred embodiment,the host cell is a gram-positive host cell that has a deletion ormutation in the naturally occurring cysteine protease said mutationresulting in deletion or inactivation of the production by the host cellof the proteolytic cysteine protease gene product. The host cell mayadditionally be genetically engineered to produced a desired protein orpolypeptide.

[0030] It may also be desired to genetically engineer host cells of anytype to produce a gram-positive cysteine protease. Such host cells areused in large scale fermentation to produce large quantities of thecysteine protease which may be isolated or purified and used in cleaningproducts, such as detergents.

[0031] I. Cysteine Protease Sequences

[0032] The CP1, CP2 and CP3 polynucleotides having the sequences asshown in FIGS. 1A-1B, 5A-5B and 6A-6B, respectively, encode the Bacillussubtilis cysteine proteases CP1, CP2 and CP3. As will be understood bythe skilled artisan, due to the degeneracy of the genetic code, avariety of polynucleotides can encode the Bacillus subtilis CP1, CP2 andCP3. The present invention encompasses all such polynucleotides.

[0033] The present invention encompasses CP1, CP2 and CP3 polynucleotidehomologs encoding gram-positive microorganism cysteine proteases CP1,CP2 and CP3, respectively, which have at least 80%, or at least 90% orat least 95% identity to B. subtilis CP1, CP2 and CP3 as long as thehomolog encodes a protein that has proteolytic activity.

[0034] Gram-positive polynucleotide homologs of B. subtilis CP1, CP2 orCP3 may be obtained by standard procedures known in the art from, forexample, cloned DNA (e.g., a DNA “library”), genomic DNA libraries, bychemical synthesis once identified, by cDNA cloning, or by the cloningof genomic DNA, or fragments thereof, purified from a desired cell.(See, for example, Sambrook et al., 1989, Molecular Cloning, ALaboratory Manual, 2d Ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.; Glover, D. M. (ed.), 1985, DNA Cloning: A PracticalApproach, MRL Press, Ltd., Oxford, U.K. Vol. I, II.) A preferred sourceis from genomic DNA. Nucleic acid sequences derived from genomic DNA maycontain regulatory regions in addition to coding regions. Whatever thesource, the isolated CP1, CP2 or CP3 gene should be molecularly clonedinto a suitable vector for propagation of the gene.

[0035] In the molecular cloning of the gene from genomic DNA, DNAfragments are generated, some of which will encode the desired gene. TheDNA may be cleaved at specific sites using various restriction enzymes.Alternatively, one may use DNAse in the presence of manganese tofragment the DNA, or the DNA can be physically sheared, as for example,by sonication. The linear DNA fragments can then be separated accordingto size by standard techniques, including but not limited to, agaroseand polyacrylamide gel electrophoresis and column chromatography.

[0036] Once the DNA fragments are generated, identification of thespecific DNA fragment containing the CP1, CP2 or CP3 may be accomplishedin a number of ways. For example, a B. subtilis CP1, CP2 or CP3 gene ofthe present invention or its specific RNA, or a fragment thereof, suchas a probe or primer, may be isolated and labeled and then used inhybridization assays to detect a gram-positive CP1, CP2 or CP3 gene.(Benton, W. and Davis, R., 1977, Science 196:180; Grunstein, M. AndHogness, D., 1975, Proc. Natl. Acad. Sci. USA 72:3961). Those DNAfragments sharing substantial sequence similarity to the probe willhybridize under stringent conditions.

[0037] Accordingly, the present invention provides a method for thedetection of gram-positive CP1, CP2 and CP3 polynucleotide homologswhich comprises hybridizing part or all of a nucleic acid sequence of B.subtilis CP1, CP2 and CP3 with gram-positive microorganism nucleic acidof either genomic or cDNA origin.

[0038] Also included within the scope of the present invention aregram-positive microorganism polynucleotide sequences that are capable ofhybridizing to the nucleotide sequence of B. subtilis CP1, CP2 or CP3under conditions of intermediate to maximal stringency. Hybridizationconditions are based on the melting temperature (Tm) of the nucleic acidbinding complex, as taught in Berger and Kimmel (1987, Guide toMolecular Cloning Techniques, Methods in Enzymology, Vol 152. AcademicPress, San Diego Calif.) incorporated herein by reference, and confer adefined “stringency” as explained below.

[0039] “Maximum stringency” typically occurs at about Tm −5° C. (5° C.below the Tm of the probe); “high stringency” at about 5° C. to 10° C.below Tm; “intermediate stringency” at about 10° C. to 20° C. below Tm;and “low stringency” at about 20° C. to 25° C. below Tm. As will beunderstood by those of skill in the art, a maximum stringencyhybridization can be used to identify or detect identical polynucleotidesequences while an intermediate or low stringency hybridization can beused to identify or detect polynucleotide sequence homologs.

[0040] The term “hybridization” as used herein shall include “theprocess by which a strand of nucleic acid joins with a complementarystrand through base pairing” (Coombs J. (1994) Dictionary ofBiotechnology, Stockton Press, New York N.Y.).

[0041] The process of amplification as carried out in polymerase chainreaction (PCR) technologies is described in Dieffenbach C W and G SDveksler (1995, PCR Primer, a Laboratory Manual, Cold Spring HarborPress, Plainview N.Y.). A nucleic acid sequence of at least about 10nucleotides and as many as about 60 nucleotides from B. subtilis CP1,CP2 or CP3 preferably about 12 to 30 nucleotides, and more preferablyabout 20-25 nucleotides can be used as a probe or PCR primer.

[0042] The B. subtilis amino acid sequences CP1, CP2 and CP3 (shown inFIGS. 2, 4 and 3, respectively) were identified via a FASTA search ofBacillus subtilis genomic nucleic acid sequences. B. subtilis CP1 (YJDE)was identified by its structural homology to the cysteine proteasepapain having the sequence designated “papa_carpa.p”. As shown in FIG.2, YJDE has the motif GXCWAF as well as the conserved catalytic residuesHis/Ala and Asn/Ser. CP2 (YdHS) and CP3 (PMI) were identified upon theirstructural homology to CP1 (YJDE). The presence of GXCWAF as well asresidues His/Ala and Asn/Ser is noted in FIGS. 3 and 4. CP3 (PMI) waspreviously characterized as a possible phosphomannose isomerase,(Noramata). There has been no previous characterization of CP3 as acysteine protease.

[0043] II. Expression Systems

[0044] The present invention provides host cells, expression methods andsystems for the enhanced production and secretion of desiredheterologous or homologous proteins in gram-positive microorganisms. Inone embodiment, a host cell is genetically engineered to have a deletionor mutation in the gene encoding a gram-positive CP1, CP2 or CP3 suchthat the respective activity is deleted. In another embodiment of thepresent invention, a gram-positive microorganism is geneticallyengineered to produce a cysteine protease of the present invention.

[0045] Inactivation of a Gram-Positive Cysteine Protease in a Host Cell

[0046] Producing an expression host cell incapable of producing thenaturally. occurring cysteine protease necessitates the replacementand/or inactivation of the naturally occurring gene from the genome ofthe host cell. In a preferred embodiment, the mutation is anon-reverting mutation.

[0047] One method for mutating nucleic acid encoding a gram-positivecysteine protease is to clone the nucleic acid or part thereof, modifythe nucleic acid by site directed mutagenesis and reintroduce themutated nucleic acid into the cell on a plasmid. By homologousrecombination, the mutated gene may be introduced into the chromosome.In the parent host cell, the result is that the naturally occurringnucleic acid and the mutated nucleic acid are located in tandem on thechromosome. After a second recombination, the modified sequence is leftin the chromosome having thereby effectively introduced the mutationinto the chromosomal gene for progeny of the parent host cell.

[0048] Another method for inactivating the cysteine protease proteolyticactivity is through deleting the chromosomal gene copy. In a preferredembodiment, the entire gene is deleted, the deletion occurring in suchas way as to make reversion impossible. In another preferred embodiment,a partial deletion is produced, provided that the nucleic acid sequenceleft in the chromosome is too short for homologous recombination with aplasmid encoded cysteine protease gene. In another preferred embodiment,nucleic acid encoding the catalytic amino acid residues are deleted.

[0049] Deletion of the naturally occurring gram-positive microorganismcysteine protease can be carried out as follows. A cysteine proteasegene including its 5′ and 3′ regions is isolated and inserted into acloning vector. The coding region of the cysteine protease gene isdeleted form the vector in vitro, leaving behind a sufficient amount ofthe 5′ and 3′ flanking sequences to provide for homologous recombinationwith the naturally occurring gene in the parent host cell. The vector isthen transformed into the gram-positive host cell. The vector integratesinto the chromosome via homologous recombination in the flankingregions. This method leads to a gram-positive strain in which theprotease gene has been deleted.

[0050] The vector used in an integration method is preferably a plasmid.A selectable marker may be included to allow for ease of identificationof desired recombinant microorgansims. Additionally, as will beappreciated by one of skill in the art, the vector is preferably onewhich can be selectively integrated into the chromosome. This can beachieved by introducing an inducible origin of replication, for example,a temperature sensitive origin into the plasmid. By growing thetransformants at a temperature to which the origin of replication issensitive, the replication function of the plasmid is inactivated,thereby providing a means for selection of chromosomal integrants.Integrants may be selected for growth at high temperatures in thepresence of the selectable marker, such as an antibiotic. Integrationmechanisms are described in WO 88/06623.

[0051] Integration by the Campbell-type mechanism can take place in the5′ flanking region of the protease gene, resulting in a proteasepositive strain carrying the entire plasmid vector in the chromosome inthe cysteine protease locus. Since illegitimate recombination will givedifferent results it will be necessary to determine whether the completegene has been deleted, such as through nucleic acid sequencing orrestriction maps.

[0052] Another method of inactivating the naturally occurring cysteineprotease gene is to mutagenize the chromosomal gene copy by transforminga gram-positive microorganism with oligonucleotides which are mutagenic.Alternatively, the chromosomal cysteine protease gene can be replacedwith a mutant gene by homologous recombination.

[0053] The present invention encompasses host cells having additionalprotease deletions or mutations, such as deletions or mutations in apr,npr, epr, mpr and others known to those of skill in the art.

[0054] One assay for the detection of mutants involves growing theBacillus host cell on medium containing a protease substrate andmeasuring the appearance or lack thereof, of a zone of clearing or haloaround the colonies. Host cells which have an inactive protease willexhibit little or no halo around the colonies.

[0055] III. Production of Cysteine Protease

[0056] For production of cysteine protease in a host cell, an expressionvector comprising at least one copy of nucleic acid encoding agram-positive microorganism CP1, CP2 or CP3, and preferably comprisingmultiple copies, is transformed into the host cell under conditionssuitable for expression of the cysteine protease. In accordance with thepresent invention, polynucleotides which encode a gram-positivemicroorganism CP1, CP2 or CP3, or fragments thereof, or fusion proteinsor polynucleotide homolog sequences that encode amino acid variants ofB. subtilis CP1, CP2 or CP3, may be used to generate recombinant DNAmolecules that direct their expression in host cells. In a preferredembodiment, the gram-positive host cell belongs to the genus Bacillus.In another preferred embodiment, the gram positive host cell is B.subtilis.

[0057] As will be understood by those of skill in the art, it may beadvantageous to produce polynucleotide sequences possessingnon-naturally occurring codons. Codons preferred by a particulargram-positive host cell (Murray E et al (1989) Nuc Acids Res 17:477-508)can be selected, for example, to increase the rate of expression or toproduce recombinant RNA transcripts having desirable properties, such asa longer half-life, than transcripts produced from naturally occurringsequence.

[0058] Altered CP1, CP2 or CP3 polynucleotide sequences which maybe usedin accordance with the invention include deletions, insertions orsubstitutions of different nucleotide residues resulting in apolynucleotide that encodes the same or a functionally equivalent CP1,CP2 or CP3 homolog, respectively. As used herein a “deletion” is definedas a change in either nucleotide or amino acid sequence in which one ormore nucleotides or amino acid residues, respectively, are absent.

[0059] As used herein an “insertion” or “addition” is that change in anucleotide or amino acid sequence which has resulted in the addition ofone or more nucleotides or amino acid-residues, respectively, ascompared to the naturally occurring CP1, CP3 or CP3.

[0060] As used herein “substitution” results from the replacement of oneor more nucleotides or amino acids by different nucleotides or aminoacids, respectively.

[0061] The encoded protein may also show deletions, insertions orsubstitutions of amino acid residues which produce a silent change andresult in a functionally CP1, CP2 or CP3 variant. Deliberate amino acidsubstitutions may be made on the basis of similarity in polarity,charge, solubility, hydrophobicity, hydrophilicity, and/or theamphipathic nature of the residues as long as the variant retains theability to modulate secretion. For example, negatively charged aminoacids include aspartic acid and glutamic acid; positively charged aminoacids include lysine and arginine; and amino acids with uncharged polarhead groups having similar hydrophilicity values include leucine,isoleucine, valine; glycine, alanine; asparagine, glutamine; serine,threonine, phenylalanine, and tyrosine.

[0062] The CP1, CP2 or CP3 polynucleotides of the present invention maybe engineered in order to modify the cloning, processing and/orexpression of the gene product. For example, mutations may be introducedusing techniques which are well known in the art, eg, site-directedmutagenesis to insert new restriction sites, to alter glycosylationpatterns or to change codon preference, for example.

[0063] In one embodiment of the present invention, a gram-positivemicroorganism CP1, CP2 or CP3 polynucleotide may be ligated to aheterologous sequence to encode a fusion protein. A fusion protein mayalso be engineered to contain a cleavage site located between thecysteine protease nucleotide sequence and the heterologous proteinsequence, so that the cysteine protease may be cleaved and purified awayfrom the heterologous moiety.

[0064] IV. Vector Sequences

[0065] Expression vectors used in expressing the cysteine proteases ofthe present invention in gram-positive microorganisms comprise at leastone promoter associated with a cysteine protease selected from the groupconsisting of CP1, CP2 and CP3, which promoter is functional in the hostcell. In one embodiment of the present invention, the promoter is thewild-type promoter for the selected cysteine protease and in anotherembodiment of the present invention, the promoter is heterologous to thecysteine protease, but still functional in the host cell. In onepreferred embodiment of the present invention, nucleic acid encoding thecysteine protease is stably integrated into the microorganism genome.

[0066] In a preferred embodiment, the expression vector contains amultiple cloning site cassette which preferably comprises at least onerestriction endonuclease site unique to the vector, to facilitate easeof nucleic acid manipulation. In a preferred embodiment, the vector alsocomprises one or more selectable markers. As used herein, the termselectable marker refers to a gene capable of expression in thegram-positive host which allows for ease of selection of those hostscontaining the vector. Examples of such selectable markers include butare not limited to antibiotics, such as, erythromycin, actinomycin,chloramphenicol and tetracycline.

[0067] V. Transformation

[0068] A variety of host cells can be used for the production of CP1,CP2 and CP3 including bacterial, fungal, mammalian and insects cells.General transformation procedures are taught in Current Protocols InMolecular Biology (vol. 1, edited by Ausubel et al., John Wiley & Sons,Inc. 1987, Chapter 9) and include calcium phosphate methods,transformation using DEAE-Dextran and electroporation. Planttransformation methods are taught in Rodriquez (WO 95/14099, published26 May 1995).

[0069] In a preferred embodiment, the host cell is a gram-positivemicroorganism and in another preferred embodiment, the host cell isBacillus. In one embodiment of the present invention, nucleic acidencoding one or more cysteine protease(s) of the present invention isintroduced into a host cell via an expression vector capable ofreplicating within the Bacillus host cell. Suitable replicating plasmidsfor Bacillus are described in Molecular Biological Methods for Bacillus,Ed. Harwood and Cutting, John Wiley & Sons, 1990, hereby expresslyincorporated by reference; see chapter 3 on plasmids. Suitablereplicating plasmids for B. subtilis are listed on page 92.

[0070] In another embodiment, nucleic acid encoding a cysteineprotease(s) of the present invention is stably integrated into themicroorganism genome. Preferred host cells are gram-positive host cells.Another preferred host is Bacillus. Another preferred host is Bacillussubtilis. Several strategies have been described in the literature forthe direct cloning of DNA in Bacillus. Plasmid marker rescuetransformation involves the uptake of a donor plasmid by competent cellscarrying a partially homologous resident plasmid (Contente et al.,Plasmid 2:555-571(1979); Haima et al., Mol. Gen. Genet. 223:185-191(1990); Weinrauch et al., J. Bacteriol. 154(3):1077-1087 (1983); andWeinrauch et al., J. Bacteriol. 169(3):1205-1211 (1987)). The incomingdonor plasmid recombines with the homologous region of the resident“helper” plasmid in a process that mimics chromosomal transformation.

[0071] Transformation by protoplast transformation is described for B.subtilis in Chang and Cohen, (1979) Mol. Gen. Genet 168:111-115; for B.megaterium in Vorobjeva et al., (1980) FEMS Microbiol. Letters7:261-263; for B. amyloliquefaciens in Smith et al., (1986) Appl. andEnv. Microbiol. 51:634; for B. thuringiensis in Fisher et al., (1981)Arch. Microbiol. 139:213-217; for B. sphaericus in McDonald (1984) J.Gen. Microbiol. 130:203; and B. larvae in Bakhiet et al., (1985) 49:577.Mann et al., (1986, Current Microbiol. 13:131-135) report ontransformation of Bacillus protoplasts and Holubova, (1985) FoliaMicrobiol. 30:97) disclose methods for introducing DNA into protoplastsusing DNA containing liposomes.

[0072] VI. Identification of Transformants

[0073] Whether a host cell has been transformed with a mutated or anaturally occurring gene encoding a gram-positive CP1, CP2 or CP3,detection of the presence/absence of marker gene expression can suggestswhether the gene of interest is present However, its expression shouldbe confirmed. For example, if the nucleic acid encoding a cysteineprotease is inserted within a marker gene sequence, recombinant cellscontaining the insert can be identified by the absence of marker genefunction. Alternatively, a marker gene can be placed in tandem withnucleic acid encoding the cysteine protease under the control of asingle promoter. Expression of the marker gene in response to inductionor selection usually indicates expression of the cysteine protease aswell.

[0074] Alternatively, host cells which contain the coding sequence for acysteine protease and express the protein may be identified by a varietyof procedures known to those of skill in the art. These proceduresinclude, but are not limited to, DNA-DNA or DNA-RNA hybridization andprotein bioassay or immunoassay techniques which include membrane-based,solution-based, or chip-based technologies for the detection and/orquantification of the nucleic acid or protein.

[0075] The presence of the cysteine polynucleotide sequence can bedetected by DNA-DNA or DNA-RNA hybridization or amplification usingprobes, portions or fragments of B. subtilis CP1, CP2 or CP3.

[0076] VII Assay of Protease Activity

[0077] There are various assays known to those of skill in the art fordetecting and measuring protease activity. There are assays based uponthe release of acid-soluble peptides from casein or hemoglobin measuredas absorbance at 280 nm or colorimetrically using the Folin method(Bergrneyer, et al., 1984, Methods of Enzymatic Analysis vol. 5,Peptidases, Proteinases and their Inhibitors, Verlag Chemie, Weinheim).Other assays involve the solubilization of chromogenic substrates (Ward,1983, Proteinases, in Microbial Enzymes and Biotechnology (W. M.Fogarty, ed.), Applied Science, London, pp. 251-317).

[0078] VIII Secretion of Recombinant Proteins

[0079] Means for determining the levels of secretion of a heterologousor homologous protein in a gram-positive host cell and detectingsecreted proteins include, using either polyclonal or monoclonalantibodies specific for the protein. Examples include enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescentactivated cell sorting (FACS). These and other assays are described,among other places, in Hampton R et al (1990, Serological Methods, aLaboratory Manual, APS Press, St Paul Minn.) and Maddox D E et al (1983,J Exp Med 158:1211).

[0080] A wide variety of labels and conjugation techniques are known bythose skilled in the art and can be used in various nucleic and aminoacid assays. Means for producing labeled hybridization or PCR probes fordetecting specific polynucleotide sequences include oligolabeling, nicktranslation, end-labeling or PCR amplification using a labelednucleotide. Alternatively, the nucleotide sequence, or any portion ofit, may be cloned into a vector for the production of an mRNA probe.Such vectors are known in the art, are commercially available, and maybe used to synthesize RNA probes in vitro by addition of an appropriateRNA polymerase such as T7, T3 or SP6 and labeled nucleotides.

[0081] A number of companies such as Pharmacia Biotech (PiscatawayN.J.), Promega (Madison Wis.), and US Biochemical Corp (Cleveland Ohio)supply commercial kits and protocols for these procedures. Suitablereporter molecules or labels include those radionuclides, enzymes,fluorescent, chemiluminescent, or chromogenic agents as well assubstrates, cofactors, inhibitors, magnetic particles and the like.Patents teaching the use of such labels include U.S. Pat. Nos.3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and4,366,241. Also, recombinant immunoglobulins may be produced as shown inU.S. Pat. No. 4,816,567 and incorporated herein by reference.

[0082] IX Purification of Proteins

[0083] Gram positive host cells transformed with polynucleotidesequences encoding heterologous or homologous protein may be culturedunder conditions suitable for the expression and recovery of the encodedprotein from cell culture. The protein produced by a recombinantgram-positive host cell comprising a mutation or deletion of thecysteine protease activity will be secreted into the culture media.Other recombinant constructions may join the heterologous or homologouspolynucleotide sequences to nucleotide sequence encoding a polypeptidedomain which will facilitate purification of soluble proteins (Kroll D Jet al (1993) DNA Cell Biol 12:441-53).

[0084] Such purification facilitating domains include, but are notlimited to, metal chelating peptides such as histidine-tryptophanmodules that allow purification on immobilized metals (Porath J (1992)Protein Expr Purif 3:263-281), protein A domains that allow purificationon immobilized immunoglobulin, and the domain utilized in the FLAGSextension/affinity purification system (Immunex Corp, Seattle Wash.).The inclusion of a cleavable linker sequence such as Factor XA orenterokinase (Invitrogen, San Diego Calif.) between the purificationdomain and the heterologous protein can be used to facilitatepurification.

[0085] X Uses of the Present Invention

[0086] CP1, CP2 and CP3 and Genetically Engineered Host Cells

[0087] The present invention provides genetically engineered host cellscomprising preferably non-revertable mutations or deletions in thenaturally occurring gene encoding CP1, CP2 or CP3 such that theproteolytic activity is diminished or deleted altogether. The host cellmay contain additional protease deletions, such as deletions of themature subtilisn protease and/or mature neutral protease disclosed inU.S. Pat. No. 5,264,366.

[0088] In a preferred embodiment, the host cell is further geneticallyengineered to produce a desired protein or polypeptide. In a preferredembodiment the host cell is a Bacillus. In another preferred embodiment,the host cell is a Bacillus subtilis.

[0089] In an alternative embodiment, a host cell is geneticallyengineered to produce a gram-positive CP1, CP2 or CP3. In a preferredembodiment, the host cell is grown under large scale fermentationconditions, the CP1, CP2 or CP3 is isolated and/or purified and used incleaning compositions such as detergents. WO 95/10615 disclosesdetergent formulation.

[0090] CP1, CP2 and CP3 Polynucleotides

[0091] A B. subtlis polynucleotide, or any part thereof, provides thebasis for detecting the presence of gram-positive microorganismpolynucleotide homologs through hybridization techniques and PCRtechnology.

[0092] Accordingly, one aspect of the present invention is to providefor nucleic acid hybridization and PCR probes which can be used todetect polynucleotide sequences, including genomic and cDNA sequences,encoding gram-positive CP1, CP2 or CP3 or portions thereof.

[0093] The manner and method of carrying out the present invention maybe more fully understood by those of skill in the art by reference tothe following examples, which examples are not intended in any manner tolimit the scope of the present invention or of the claims directedthereto

EXAMPLE I Preparation of a Genomic Library

[0094] The following example illustrates the preparation of a Bacillusgenomic library.

[0095] Genomic DNA from Bacillus cells is prepared as taught in CurrentProtocols In Molecular Biology vol. 1, edited by Ausubel et al., JohnWiley & Sons, Inc. 1987, chapter 2.4.1. Generally, Bacillus cells from asaturated liquid culture are lysed and the proteins removed by digestionwith proteinase K. Cell wall debris, polysaccharides, and remainingproteins are removed by selective precipitation with CTAB, and highmolecular weight genomic DNA is recovered from the resulting supernatantby isopropanol precipitation. If exceptionally clean genomic DNA isdesired, an additional step of purifying the Bacillus genomic DNA on acesium chloride gradient is added.

[0096] After obtaining purified genomic DNA, the DNA is subjected toSau3A digestion. Sau3A recognizes the 4 base pair site GATC andgenerates fragments compatible with several convenient phage lambda andcosmid vectors. The DNA is subjected to partial digestion to increasethe chance of obtaining random fragments.

[0097] The partially digested Bacillus genomic DNA is subjected to sizefractionation on a 1% agarose gel prior to cloning into a vector.Alternatively, size fractionation on a sucrose gradient can be used. Thegenomic DNA obtained from the size fractionation step is purified awayfrom the agarose and ligated into a cloning vector appropriate for usein a host cell and transformed into the host cell.

EXAMPLE II Detection of Gram-Postive Microorganisms

[0098] The following example describes the detection of gram-positivemicroorganism CP1. The same procedures can be used to detect CP2 andCP3.

[0099] DNA derived from a gram-positive microorganism is preparedaccording to the methods disclosed in Current Protocols in MolecularBiology, Chap. 2 or 3. The nucleic acid is subjected to hybridizationand/or PCR amplification with a probe or primer derived from CP1. Apreferred probe comprises the nucleic acid section containing theconserved motif GXCWAF.

[0100] The nucleic acid probe is labeled by combining 50 pmol of thenucleic acid and 250 mCi of [gamma ³²P] adenosine triphosphate(Amersham, Chicago Ill.) and T4 polynucleotide kinase (DuPont NEN®,Boston Mass.). The labeled probe is purified with Sephadex G-25 superfine resin column (Pharmacia). A portion containing 10⁷ counts perminute of each is used in a typical membrane based hybridizationanalysis of nucleic acid sample of either genomic or cDNA origin.

[0101] The DNA sample which has been subjected to restrictionendonuclease digestion is fractionated on a 0.7 percent agarose gel andtransferred to nylon membranes (Nytran Plus, Schleicher & Schuell,Durham N.H.). Hybridization is carried out for 16 hours at 40 degrees C.To remove nonspecific signals, blots are sequentially washed at roomtemperature under increasingly stringent conditions up to 0.1× salinesodium citrate and 0.5% sodium dodecyl sulfate. The blots are exposed tofilm for several hours, the film developed and hybridization patternsare compared visually to detect polynucleotide homologs of B. subtilisCP1. The homologs are subjected to confirmatory nucleic acid sequencing.Methods for nucleic acid sequencing are well known in the art.Conventional enzymatic methods employ DNA polymerase Klenow fragment,SEQUENASE® (US Biochemical Corp, Cleveland, Ohio) or Taq polymerase toextend DNA chains from an oligonucleotide primer annealed to the DNAtemplate of interest.

[0102] Various other examples and modifications of the foregoingdescription and examples will be apparent to a person skilled in the artafter reading the disclosure without departing from the spirit and scopeof the invention, and it is intended that all such examples ormodifications be included within the scope of the appended claims. Allpublications and patents referenced herein are hereby incorporated byreference in their entirety.

1 7 1 945 DNA Bacillius subtilis 1 atgacgactg aaccgttatt tttcaagcctgttttcaaag aaagaatttg gggcgggacc 60 gctttagctg attttggcta taccattccgtcacaacgaa caggggagtg ctgggctttt 120 gccgcgcatc aaaatggtca aagcgttgttcaaaacggaa tgtataaggg gttcacgctc 180 agcgaattat gggaacatca cagacatttattcggacagc ttgaagggga ccgtttccct 240 ctgcttacaa aaatattaga tgctgaccaggacttatctg ttcaggtgca tccgaatgat 300 gaatatgcca acatacatga aaacggtgagcttggaaaaa cagaatgctg gtacattatt 360 gattgccaaa aagatgccga gattatttatggccacaatg caacaacaaa ggaagaacta 420 actaccatga tagagcgtgg agaatgggatgagctcttgc gccgtgtaaa ggtaaagccg 480 ggggattttt tctatgtgcc aagcggtactgttcatgcga ttggaaaagg aattcttgct 540 ttggagacgc agcagaactc agacacaacctacagattat atgattatga ccgaaaagat 600 gcagaaggca agctgcgcga gcttcatctgaaaaagagca ttgaagtgat agaggtcccg 660 tctattccag aacggcatac agttcaccatgaacaaattg aggatttgct tacaacgaca 720 ttgattgaat gcgcttactt ttcggtggggaaatggaact tatcaggatc agcaagctta 780 aagcagcaaa aaccattcct tcttatcagtgtgattgaag gggagggccg tatgatctct 840 ggtgagtatg tctatccttt caaaaaaggagatcatatgt tgctgcctta cggtcttgga 900 gaatttaaac tcgaaggata tgcagaatgtatcgtctccc atctg 945 2 315 PRT Bacillus subtilis 2 Met Thr Thr Glu ProLeu Phe Phe Lys Pro Val Phe Lys Glu Arg Ile 1 5 10 15 Trp Gly Gly ThrAla Leu Ala Asp Phe Gly Tyr Thr Ile Pro Ser Gln 20 25 30 Arg Thr Gly GluCys Trp Ala Phe Ala Ala His Gln Asn Gly Gln Ser 35 40 45 Val Val Gln AsnGly Met Tyr Lys Gly Phe Thr Leu Ser Glu Leu Trp 50 55 60 Glu His His ArgHis Leu Phe Gly Gln Leu Glu Gly Asp Arg Phe Pro 65 70 75 80 Leu Leu ThrLys Ile Leu Asp Ala Asp Gln Asp Leu Ser Val Gln Val 85 90 95 His Pro AsnAsp Glu Tyr Ala Asn Ile His Glu Asn Gly Glu Leu Gly 100 105 110 Lys ThrGlu Cys Trp Tyr Ile Ile Asp Cys Gln Lys Asp Ala Glu Ile 115 120 125 IleTyr Gly His Asn Ala Thr Thr Lys Glu Glu Leu Thr Thr Met Ile 130 135 140Glu Arg Gly Glu Trp Asp Glu Leu Leu Arg Arg Val Lys Val Lys Pro 145 150155 160 Gly Asp Phe Phe Tyr Val Pro Ser Gly Thr Val His Ala Ile Gly Lys165 170 175 Gly Ile Leu Ala Leu Glu Thr Gln Gln Asn Ser Asp Thr Thr TyrArg 180 185 190 Leu Tyr Asp Tyr Asp Arg Lys Asp Ala Glu Gly Lys Leu ArgGlu Leu 195 200 205 His Leu Lys Lys Ser Ile Glu Val Ile Glu Val Pro SerIle Pro Glu 210 215 220 Arg His Thr Val His His Glu Gln Ile Glu Asp LeuLeu Thr Thr Thr 225 230 235 240 Leu Ile Glu Cys Ala Tyr Phe Ser Val GlyLys Trp Asn Leu Ser Gly 245 250 255 Ser Ala Ser Leu Lys Gln Gln Lys ProPhe Leu Leu Ile Ser Val Ile 260 265 270 Glu Gly Glu Gly Arg Met Ile SerGly Glu Tyr Val Tyr Pro Phe Lys 275 280 285 Lys Gly Asp His Met Leu LeuPro Tyr Gly Leu Gly Glu Phe Lys Leu 290 295 300 Glu Gly Tyr Ala Glu CysIle Val Ser His Leu 305 310 315 3 220 PRT Bacillus subtilis 3 Val LeuAsn Asp Gly Asp Val Asn Ile Pro Glu Tyr Val Asp Trp Arg 1 5 10 15 GlnLys Gly Ala Val Thr Pro Val Lys Asn Gln Gly Ser Cys Gly Ser 20 25 30 CysTrp Ala Phe Ser Ala Val Val Thr Ile Glu Gly Ile Ile Lys Ile 35 40 45 ArgThr Gly Asn Leu Asn Glu Tyr Ser Glu Gln Glu Leu Leu Asp Cys 50 55 60 AspArg Arg Ser Tyr Gly Cys Asn Gly Gly Tyr Pro Trp Ser Ala Leu 65 70 75 80Gln Leu Val Ala Gln Tyr Gly Ile His Tyr Arg Asn Thr Tyr Pro Tyr 85 90 95Glu Gly Val Gln Arg Tyr Cys Arg Ser Arg Glu Lys Gly Pro Tyr Ala 100 105110 Ala Lys Thr Asp Gly Val Arg Gln Val Gln Pro Tyr Asn Glu Gly Ala 115120 125 Leu Leu Tyr Ser Ile Ala Asn Gln Pro Val Ser Val Val Leu Glu Ala130 135 140 Ala Gly Lys Asp Phe Gln Leu Tyr Arg Gly Gly Ile Phe Val GlyPro 145 150 155 160 Cys Gly Asn Lys Val Asp His Ala Val Ala Ala Val GlyTyr Gly Pro 165 170 175 Asn Tyr Ile Leu Ile Lys Asn Ser Trp Gly Thr GlyTrp Gly Glu Asn 180 185 190 Gly Tyr Ile Arg Ile Lys Arg Gly Thr Gly AsnSer Tyr Gly Val Cys 195 200 205 Gly Leu Tyr Thr Ser Ser Phe Tyr Pro ValLys Asn 210 215 220 4 948 DNA Bacillus subtilis 4 atgacgcaat caccgatttttctaacgcct gtgtttaaag aaaaaatctg gggcggaacc 60 gctttacgag atagatttggatacagtatt ccttcagaat caacggggga atgctgggcc 120 atttccgctc atccaaaaggaccgagcact gttgcaaatg gcccgtataa aggaaagaca 180 ttgatcgagc tttgggaagagcaccgtgaa gtattcggcg gcgtagaggg ggatcggttt 240 ccgcttctga caaagctgctggatgtgaag gaagatacgt caattaaagt tcaccctgat 300 gattactatg ccggagaaaacgaagaggga gaactcggca agacggaatg ctggtacatt 360 atcgactgta aggaaaacgcagaaatcatt tacgggcata cggcccgctc aaaaaccgaa 420 cttgtcacaa tgatcaacagcggtgactgg gagggcctgc tgcgaagaat caaaattaaa 480 ccgggtgatt tctattatgtgccgagcgga acgctgcacg cattgtgcaa gggggccctt 540 gttttagaga ctcagcaaaattcagatgcc acataccggg tgtacgatta tgaccgtctt 600 gatagcaacg gaagtccgagagagcttcat tttgccaaag cggtcaatgc cgccacggtt 660 ccccatgtgg acgggtatatagatgaatcg acagaatcaa gaaaaggaat aaccattaaa 720 acatttgtcc aaggggaatatttttcggtt tataaatggg acatcaatgg cgaagctgaa 780 atggctcagg atgaatcctttctgatttgc agcgtgatag aaggaagcgg tttgctcaag 840 tatgaggaca aaacatgtccgctcaaaaaa ggtgatcact ttattttgcc ggctcaaatg 900 cccgatttta cgataaaaggaacttgtacc cttatcgtgt ctcatatt 948 5 316 PRT Bacillus subtilis 5 Met ThrGln Ser Pro Ile Phe Leu Thr Pro Val Phe Lys Glu Lys Ile 1 5 10 15 TrpGly Gly Thr Ala Leu Arg Asp Arg Phe Gly Tyr Ser Ile Pro Ser 20 25 30 GluSer Thr Gly Glu Cys Trp Ala Ile Ser Ala His Pro Lys Gly Pro 35 40 45 SerThr Val Ala Asn Gly Pro Tyr Lys Gly Lys Thr Leu Ile Glu Leu 50 55 60 TrpGlu Glu His Arg Glu Val Phe Gly Gly Val Glu Gly Asp Arg Phe 65 70 75 80Pro Leu Leu Thr Lys Leu Leu Asp Val Lys Glu Asp Thr Ser Ile Lys 85 90 95Val His Pro Asp Asp Tyr Tyr Ala Gly Glu Asn Glu Glu Gly Glu Leu 100 105110 Gly Lys Thr Glu Cys Trp Tyr Ile Ile Asp Cys Lys Glu Asn Ala Glu 115120 125 Ile Ile Tyr Gly His Thr Ala Arg Ser Lys Thr Glu Leu Val Thr Met130 135 140 Ile Asn Ser Gly Asp Trp Glu Gly Leu Leu Arg Arg Ile Lys IleLys 145 150 155 160 Pro Gly Asp Phe Tyr Tyr Val Pro Ser Gly Thr Leu HisAla Leu Cys 165 170 175 Lys Gly Ala Leu Val Leu Glu Thr Gln Gln Asn SerAsp Ala Thr Tyr 180 185 190 Arg Val Tyr Asp Tyr Asp Arg Leu Asp Ser AsnGly Ser Pro Arg Glu 195 200 205 Leu His Phe Ala Lys Ala Val Asn Ala AlaThr Val Pro His Val Asp 210 215 220 Gly Tyr Ile Asp Glu Ser Thr Glu SerArg Lys Gly Ile Thr Ile Lys 225 230 235 240 Thr Phe Val Gln Gly Glu TyrPhe Ser Val Tyr Lys Trp Asp Ile Asn 245 250 255 Gly Glu Ala Glu Met AlaGln Asp Glu Ser Phe Leu Ile Cys Ser Val 260 265 270 Ile Glu Gly Ser GlyLeu Leu Lys Tyr Glu Asp Lys Thr Cys Pro Leu 275 280 285 Lys Lys Gly AspHis Phe Ile Leu Pro Ala Gln Met Pro Asp Phe Thr 290 295 300 Ile Lys GlyThr Cys Thr Leu Ile Val Ser His Ile 305 310 315 6 945 DNA Bacillussubtilis 6 atgacgcatc cattattttt agagcctgtc tttaaagaaa gactatggggagggacgaag 60 cttcgtgacg cttttggcta cgcaataccc tcacaaaaaa caggtgagtgctgggccgtt 120 tctgcacatg cccatggctc gtcgtctgta aaaaatggcc cgctggcaggaaagacactt 180 gatcaagtat ggaaagatca tccagagata ttcgggtttc cggatggtaaggtgtttccg 240 ctgctggtaa agctgctgga cgccaatatg gatctctccg tgcaagtccatcctgatgat 300 gattatgcaa aactgcacga aaatggcgac cttggtaaaa cggagtgctggtatatcatt 360 gattgcaaag atgacgccga actaattttg ggacatcatg caagcacaaaggaagagttc 420 aaacaacgaa tagaaagcgg tgattggaac gggctgctga ggcgaatcaaaatcaagcca 480 ggagatttct tttatgtgcc aagcggtaca ctccatgctt tatgtaagggaacccttgtc 540 cttgaaatcc agcaaaactc tgatacaaca tatcgcgtat acgattatgaccgctgtaat 600 gaccagggcc aaaaaagaac tcttcatata gaaaaagcca tggaagtcataacgataccg 660 catatcgata aagtgcatac accggaagta aaagaagttg gtaacgctgagatcattgtt 720 tatgtgcaat cagattattt ctcagtgtac aaatggaaga ttagcggccgagctgctttt 780 ccttcatatc aaacctattt gctggggagt gttctgagcg gatcaggacgaatcataaat 840 aatggtattc agtatgaatg caatgcaggc tcacacttta ttctgcctgcgcattttgga 900 gaatttacaa tagaaggaac atgtgaattc atgatatctc atcct 945 7315 PRT Bacillus subtilis 7 Met Thr His Pro Leu Phe Leu Glu Pro Val PheLys Glu Arg Leu Trp 1 5 10 15 Gly Gly Thr Lys Leu Arg Asp Ala Phe GlyTyr Ala Ile Pro Ser Gln 20 25 30 Lys Thr Gly Glu Cys Trp Ala Val Ser AlaHis Ala His Gly Ser Ser 35 40 45 Ser Val Lys Asn Gly Pro Leu Ala Gly LysThr Leu Asp Gln Val Trp 50 55 60 Lys Asp His Pro Glu Ile Phe Gly Phe ProAsp Gly Lys Val Phe Pro 65 70 75 80 Leu Leu Val Lys Leu Leu Asp Ala AsnMet Asp Leu Ser Val Gln Val 85 90 95 His Pro Asp Asp Asp Tyr Ala Lys LeuHis Glu Asn Gly Asp Leu Gly 100 105 110 Lys Thr Glu Cys Trp Tyr Ile IleAsp Cys Lys Asp Asp Ala Glu Leu 115 120 125 Ile Leu Gly His His Ala SerThr Lys Glu Glu Phe Lys Gln Arg Ile 130 135 140 Glu Ser Gly Asp Trp AsnGly Leu Leu Arg Arg Ile Lys Ile Lys Pro 145 150 155 160 Gly Asp Phe PheTyr Val Pro Ser Gly Thr Leu His Ala Leu Cys Lys 165 170 175 Gly Thr LeuVal Leu Glu Ile Gln Gln Asn Ser Asp Thr Thr Tyr Arg 180 185 190 Val TyrAsp Tyr Asp Arg Cys Asn Asp Gln Gly Gln Lys Arg Thr Leu 195 200 205 HisIle Glu Lys Ala Met Glu Val Ile Thr Ile Pro His Ile Asp Lys 210 215 220Val His Thr Pro Glu Val Lys Glu Val Gly Asn Ala Glu Ile Ile Val 225 230235 240 Tyr Val Gln Ser Asp Tyr Phe Ser Val Tyr Lys Trp Lys Ile Ser Gly245 250 255 Arg Ala Ala Phe Pro Ser Tyr Gln Thr Tyr Leu Leu Gly Ser ValLeu 260 265 270 Ser Gly Ser Gly Arg Ile Ile Asn Asn Gly Ile Gln Tyr GluCys Asn 275 280 285 Ala Gly Ser His Phe Ile Leu Pro Ala His Phe Gly GluPhe Thr Ile 290 295 300 Glu Gly Thr Cys Glu Phe Met Ile Ser His Pro 305310 315

We claim:
 1. An isolated polynucleotide encoding CP1 from a grampositive microorganism.
 2. The polynucleotide of claim 1 wherein CP1 hasthe amino acid sequence shown in FIGS. 1A-1B.
 3. An isolated CP1encoding nucleic acid having the nucleic acid sequence as shown inFIG.
 1. 4. An isolated CP1 from a gram-positive microorganism.
 5. Theisolated CP1 of claim 4 having the amino acid sequence as shown in FIGS.1A-1B.
 6. An isolated polynucleotide encoding CP2 from a gram positivemicroorganism.
 7. The polynucleotide of claim 6 wherein CP2 has theamino acid sequence shown in FIGS. 5A-5B.
 8. The isolated CP2 encodingnucleic acid having the sequence as shown in FIGS. 5A-5B.
 9. An isolatedCP2 from a gram-positive microorganism.
 10. The isolated CP2 of claim 9having the amino acid sequence as shown in FIGS. 5A-5B.
 11. Agram-positive microorganism having a mutation or deletion of part or allof the gene encoding CP1 said mutation or deletion resulting in theinactivation of the CP1 proteolytic activity.
 12. A gram-positivemicroorganism having a mutation or deletion of part or all of the geneencoding CP2 said mutation or deletion resulting in the inactivation ofthe CP2 proteolytic activity.
 13. A gram-positive microorganism having amutation or deletion of part or all of the gene encoding CP3 saidmutation or deletion resulting in the inactivation of the CP3proteolytic activity.
 14. The gram-positive microorganism according toclaims 11, 12 or 13 that is a member of the family Bacillus.
 15. Themicroorganism according to claim 14 wherein the member is selected fromthe group consisting of B. licheniformis, B. lentus, B. brevis, B.stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. coagulans,B. circulans, B. lautus and Bacillus thuringiensis.
 16. Themicroorganism of claim 11, 12 or 13 wherein said microorganism iscapable of expressing a heterologous protein.
 17. The host cell of claim16 wherein said heterologous protein is selected from the groupconsisting of hormone, enzyme, growth factor and cytokine.
 18. The hostcell of claim 17 wherein said heterologous protein is an enzyme.
 19. Thehost cell of claim 15 wherein said enzyme is selected from the groupconsisting of a proteases, carbohydrases, and lipases; isomerases suchas racemases, epimerases, tautomerases, or mutases; transferases,kinases and phophatases.
 20. A cleaning composition comprising acysteine protease selected from the group consisting of CP1, CP2 andCP3.
 21. An expression vector comprising nucleic acid encoding acysteine protease selected from the group consisting of CP1, CP2 andCP3.
 22. A host cell comprising an expression vector according to claim21.